PROJECT SUMMARY A major mechanism proposed for HDL's beneficial effect on cardiovascular disease risk is its capacity to promote cholesterol excretion through a process called reverse cholesterol transport (RCT). Elevated HDL cholesterol levels are generally correlated with reduced disease risk. However, recent trials of HDL-raising drugs failed to observe a clinical benefit. This has led to a revised HDL hypothesis where the functional capacity of HDL to promote peripheral cholesterol efflux and RCT, not HDL concentration, is deemed most important for its protective role. Cholesteryl ester transfer protein (CETP) facilitates the net movement of cholesteryl ester (CE) between plasma lipoproteins. In humans, following the conversion of tissue-derived cholesterol to CE on HDL, 70% of HDL CE is transferred by CETP to VLDL and LDL prior to being removal by the liver. Thus, CETP is a central player in the HDL-mediated cholesterol excretion pathway. We have shown in vitro that apolipoprotein F (ApoF) modulates CETP activity by inhibiting lipid transfers involving LDL but stimulating lipid transfer between HDL and VLDL. We hypothesize that ApoF operates as a metabolic switch by redirecting HDL-derived CE to VLDL instead of LDL, and that this reduces plasma LDL cholesterol levels and stimulates RCT. Here, we test this hypothesis and investigate mechanisms controlling ApoF activity. Aim 1 ? Determine the role of ApoF in defining the fate of HDL-derived CE ? With an established model of siRNA-mediated ApoF knockdown in hamsters, we will quantify how the loss of this key protein modifies the efflux of HDL CE to LDL and VLDL, measure the hepatic clearance of VLDL and LDL CE, and determine the impact of ApoF on HDL function and RCT in chow-fed and fat-fed animals. Aim 2 ? Define the mechanism that converts ApoF to its active form and quantify the effect of hyperlipidemia on ApoF concentration and its activation status ? Plasma ApoF exists in both active and inactive forms; active ApoF is bound to LDL. Hypercholesterolemic LDL is enriched in ApoF. We hypothesize that the molecular packing of LDL surface lipids controls ApoF binding. We will identify the molecular properties of LDL that correlate with enhanced ApoF binding and rigorously test their role in this process. Total and active ApoF concentrations will be quantified in hyperlipidemic human subjects to determine their possible contribution to aberrant lipoprotein levels. Aim 3 ? Determine the factors that control plasma ApoF levels ? In fat-fed animals, plasma ApoF is increased but hepatic APOF mRNA levels are decreased. To understand these discordant observa- tions, the molecular mechanisms causing this decrease in APOF mRNA will be determined, and the turnover of plasma ApoF in normolipidemic and hypercholesterolemic hamsters will be compared. We anticipate find- ing that greater association of ApoF with LDL lengthens its plasma residence time. Overall, these studies will provide novel insight into how ApoF regulates CETP activity and enhances the functional properties of HDL.